Burkholderia cepacia complex are emerging as an important group of drug resistant pathogens. The increasing number of infections in immunocompromised patients raises significant concern as antibiotic development continues to lag and our understanding of this unique pathogen still remains undeveloped. Overcoming antibiotic resistance in this genetically highly complex organism possessing multiple chromosomes is a significant medical and scientific challenge. Our main objective is to identify novel ways of overcoming -lactamase-mediated resistance in these and other Gram-negative pathogens. Our previous studies investigating Pen-like -lactamases in Burkholderia spp. lead us to postulate that a novel diazabicyclooctane (DBO) -lactamase inhibitor, avibactam, possesses the correct chemical features to efficiently inactivate PenA. Based upon this chemistry, we will show why avibactam works while clavulanate, sulbactam and tazobactam do not. In addition, strong preliminary evidence we have obtained leads us to hypothesize that the altered regulation of -lactamases (i.e., blapen and blaampC,) by LysR-type transcriptional regulators (LTTRs) (i.e., PenRA and PenRB) in B. cepacia complex is a critical determinant in -lactam resistance. To address these hypotheses, we will use our knowledge of structure-function relationships of PenA -lactamase to understand the mechanism of inactivation by avibactam and we will employ new genetic and biochemical approaches to dissect the regulation of -lactamase-mediated resistance in B. cepacia complex focusing on the PenRA and PenRB. Studying the pathway to inactivation by avibactam will entail detailed biochemical and mechanistic analyses. Unravelling the complex regulation of PenA -lactamase expression in B. cepacia complex will require us to use the lessons learned from Pseudomonas aeruginosa and Enterobacter spp. to understand the steps needed. To address these objectives, the biochemical inhibitory parameters of avibactam will be determined against PenA focusing on key active site residues (i.e., S130, E166, N170, R220, T237, and E276) and obtain a crystal structure of avibactam in the active site of PenA. The in vitro activity of the ceftazidime-avibactam will be assessed against diverse panel of clinical isolates of B. cepacia complex. Next, RNA-seq will be used to define the transcriptome of PenRA and PenRB. PenRA and PenRB from Burkholderia multivorans and Burkholderia cenocepacia, the two most clinically prevalent B. cepacia complex pathogens, will be cloned expressed, and purified. We will assess binding of transcription regulators to DNA and measure -lactamase induction. Finally, we will use site-directed mutagenesis to mutate positions 102, 103, 135, 221, and 264 of PenRA and PenRB and assess the impact of these changes on bla expression. We expect to show that this novel DBO inhibitor will inactivate PenA and that new mechanistic insights will be obtained against this carbapenemase as well as other difficult to inhibit -lactamases. We also anticipate that we will unravel the biochemical features of PenRA and PenRB that are necessary for the transcriptional regulation of bla expression in B. cepacia complex. Ultimately, our long term goal is to identify druggable sites on PenRA and PenRB. LTTRs may serve as a novel targets for therapies in Burkholderia spp. and other Gram-negatives. Achieving our objectives will impact the field by increasing the general understanding needed to identify novel antibiotic targets within problematic Gram-negatives.